Method for in-situ determination of higher fatty acid in white spirit

Abstract

The invention discloses a method for in-situ determination of higher fatty acid in Baijiu, and belongs to the technical field of Baijiu detection. In order to realize in-situ simultaneous detection of a plurality of higher fatty acids in Baijiu, the invention provides a method for determining a plurality of higher fatty acids in Baijiu based on combination of ultra-high performance liquid chromatography and a quadrupole-electrostatic field orbitrap mass spectrometer, according to the method, liquid sample introduction is carried out directly or after membrane passing, and analysis is carried out through a high-resolution liquid mass spectrometer. And performing external standard method quantification after SIM mode scanning to obtain the content of the 14 higher fatty acids in the to-be-detected sample. According to the method, tedious pretreatment and derivatization processes are avoided, the 14 trace or trace higher fatty acids in the Baijiu can be accurately quantified at the same time, accurate verification can be achieved in Luzhou-flavor Baijiu, Fen-flavor Baijiu, Maotai-flavor Baijiu and rice-flavor Baijiu, the steps are simple, and operation is convenient.

Description

Technical Field

[0001] The invention belongs to the technical field of liquor detection, and particularly relates to a method for in-situ determination of higher fatty acids in liquor. Background Art

[0002] In the field of liquor flavor science, the study of acid substances is an important link that cannot be ignored. In particular, the quantitative analysis of higher fatty acids is very necessary for a comprehensive understanding of the substances in the liquor. It is known that higher fatty acids play an important role in the coordination of the taste and taste perception of the liquor, but at present, no relevant detection standards for higher fatty acids in liquor have been issued. Different from the detection of fatty acids in food, the content of higher fatty acids in liquor is lower and not easy to be separated, so a method with high sensitivity and low quantitative limit is required for determination.

[0003] Gas chromatography and gas chromatography-tandem mass spectrometry are generally considered to be one of the common methods for detecting fatty acids and other non-volatile acids. However, due to the high boiling point of higher fatty acid compounds, derivatization agents are used when determining higher fatty acids by gas chromatography and gas chromatography-tandem mass spectrometry to derive higher fatty acid compounds into molecules with lower boiling points for easy vaporization. Therefore, the sample preparation process becomes complicated, in-situ analysis cannot be performed, and the sensitivity is limited and the detection limit is high, which increases the difficulty of accurately quantifying fatty acids in liquor.

[0004] Liquid chromatography and liquid chromatography-mass spectrometry are also commonly used for the detection of fatty acids. Short-chain fatty acids have ultraviolet absorption and can be quantified using liquid chromatography. However, the optical absorption of long-chain fatty acids is weaker and can be better quantified by derivatizing them into other ultraviolet absorbing substances or by using mass spectrometry.

[0005] CN202410787621.2 discloses a method for simultaneously determining a plurality of non-volatile organic acids, which comprises the following steps: pretreatment: taking the liquid to be tested and carrying out constant temperature nitrogen blowing to concentrate it to 1 / 5-1 / 3 of the volume, then diluting it with methanol to 1-3 times of the volume, filtering, and obtaining the sample to be tested; sample determination: using ultra-high performance liquid phase tandem high-resolution mass spectrometry to analyze the sample to be tested, and obtaining the content of the target detection object in the liquid to be tested; the target detection object includes fatty acids, phenolic acids and other non-volatile organic acids. Although this method uses liquid chromatography-mass spectrometry to analyze the non-volatile organic acids in different flavor liquors, and there are many types of detection, there are still some fatty acids that are not covered, including hydroxy fatty acids and fatty olefinic acids, etc. These compounds may have a certain effect on the taste and flavor of liquor. In addition, although this method does not involve the derivatization process, this method will cause some non-volatile organic acids in the same wine sample to not be detected due to the interference of ethanol or other volatile substances, so its sample needs to be concentrated by nitrogen blowing, and there is still inconvenience in operation.

[0006] CN202311106029.3 discloses a quantitative method for multiple non-volatile organic acids in liquor, which comprises the following steps: (1) sample pretreatment: evaporating and concentrating the liquor sample to obtain a sample to be tested; (2) sample determination: using ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry to analyze the organic acids in the sample to be tested to obtain the content of the organic acids in the liquor sample; wherein the conditions for the evaporation and concentration are: temperature of 41-49°C, rotation speed of 65-75rpm, and pressure of -0.2-0MPa; the chromatographic column used in the ultra-high performance liquid chromatography is: BEH C18 Ax (2.1×100mm, 1.7μm); the organic acid types include: at least one of succinic acid, glyoxylic acid, glutaric acid, malic acid, 2-hydroxybutyric acid, D-pantothenic acid, 2-methylsuccinic acid, p-hydroxybenzoic acid, 2-hydroxy-3-methylbutyric acid, suberic acid, azelaic acid, sebacic acid, 3-phenyllactic acid, and salicylic acid. However, this method is only applicable to volatile low-level fatty acids, and it is difficult to accurately detect multiple high-level fatty acids at the same time; in addition, this method requires evaporation and concentration, and its rotary evaporation temperature plays a key role in the analysis of extremely trace amounts of non-volatile organic acids in liquor.

[0007] CN202410676590.3 discloses a method for detecting higher fatty acid esters in wine samples by triple quadrupole gas chromatography-mass spectrometry, in which the sample to be tested can be directly injected or injected after passing through a 0.22 μm filter membrane; the higher fatty acid esters include ethyl palmitate, ethyl linoleate and ethyl oleate. Although this method can be injected directly, the detected substance is fatty acid ester; and due to the difference in properties between fatty acid esters and fatty acids, the volatility and optical absorption of fatty acids are weaker, which is not suitable for direct injection detection in gas chromatography, so this method is not suitable for detecting higher fatty acids, or it requires the acid to be derivatized into esters.

[0008] CN202211177341.7 discloses a method for simultaneously determining a variety of non-volatile flavoring substances in liquor, in which the sampling volume is 2-3 ml, the sampling is placed in a centrifuge tube for high-speed centrifugation, and then the liquor sample after centrifugation is filtered through a 0.22 μm water-based PES syringe filter membrane; the flavoring substances include at least one of organic acids, biogenic amines, and amino acids; the organic acids include: L-tartaric acid, L-malic acid, gallic acid, ferulic acid, catechin, caffeic acid, luteolin, chlorogenic acid, p-coumaric acid, 2,3-dihydroxypropionic acid, and citric acid. However, this method is only applicable to volatile low-level fatty acids, and it is difficult to accurately detect multiple high-level fatty acids at the same time. Summary of the invention

[0009] The present invention aims to provide a method for determining a variety of higher fatty acid compounds in liquor based on ultra-high performance liquid chromatography combined with a quadrupole-electrostatic field orbital trap mass spectrometer. The method avoids cumbersome pretreatment and derivatization processes through direct liquid sampling, and simultaneously realizes accurate quantification of 14 higher fatty acids including trace or trace amounts of 16-hydroxypalmitic acid, 2-hydroxyoleic acid, arachidic acid, arachidonic acid, cis-11-eicosenoic acid, cis-9-hexadecenoic acid, lauric acid, oleic acid, linoleic acid, linolenic acid, myristic acid, palmitic acid, pentadecanoic acid and stearic acid in liquor. The method has simple steps and is convenient to operate.

[0010] In order to achieve the purpose of the present invention, the present invention provides a method for quickly detecting a variety of higher fatty acid compounds in liquor based on ultra-high performance liquid chromatography combined with quadrupole-electrostatic field orbital trap mass spectrometry, using the Optimization option in the MS Tune software in the quadrupole-electrostatic field orbital trap mass spectrometer to optimize the ion source region parameters of the target higher fatty acid, including spray voltage, sheath gas, auxiliary gas, purge gas and other parameters, and monitoring the real-time response of the target compound by injection sampling to obtain the optimal parameters. According to the best quality spectrum determination conditions, moderate parameters are selected to collect compound mass spectrum data, and the higher fatty acid compounds in liquor are quantified using the SIM mode.

[0011] The present invention provides a method for simultaneously determining multiple higher fatty acid compounds in liquor without pretreatment or derivatization, comprising the following steps:

[0012] A. Preparation of standard working solution: accurately weigh 16-hydroxypalmitic acid, 2-hydroxyoleic acid, arachidic acid, arachidonic acid, cis-11-eicosenoic acid, cis-9-hexadecenoic acid, lauric acid, oleic acid, linoleic acid, linolenic acid, myristic acid, palmitic acid, pentadecanoic acid and stearic acid, dissolve in 45-55% ethanol aqueous solution to obtain mixed standard working solutions of a series of concentrations;

[0013] B. Preparation of test samples: directly use liquor as the test sample, or filter the liquor through a 0.22μm organic filter membrane and use it as the test sample;

[0014] C. Drawing of standard working curves: The mixed standard working solution was injected into an ultra-high performance liquid chromatography combined with a quadrupole-electrostatic field orbital trap mass spectrometer to extract and integrate the target ion peaks. The concentration of the target higher fatty acid was used as the abscissa and the peak area was used as the ordinate to obtain the standard working curves of 14 higher fatty acids respectively;

[0015] D. Detection of the sample to be tested: inject the sample to be tested into an ultra-high performance liquid chromatography combined with a quadrupole-electrostatic field orbital trap mass spectrometer, extract and integrate the target ion peak under the same detection conditions as in step C, obtain the peak area of ​​the target higher fatty acid in the sample to be tested, and calculate the concentration of 14 higher fatty acids in the sample to be tested according to the standard working curve obtained in step C;

[0016] The chromatographic conditions are as follows: chromatographic column: Waters ACQUITY UPLC BEH C18, 1.7 μm, 150 mm*2.1 μm; mobile phase: mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: methanol; gradient elution program:

[0017] Serial number Time (min) Flow rate (mL / min) Mobile phase A Mobile phase B Gradient Curve 1 -10 0.2 5.0 95.0 5 2 -5 0.2 5.0 95.0 5 3 -4 0.2 45.0 55.0 5 4 0 0.2 45.0 55.0 5 5 8 0.2 45.0 55.0 5 6 40 0.2 5.0 95.0 5 7 50 0.2 5.0 95.0 5 ;

[0018] The mass spectrometry conditions were as follows: scanning mode: SIM scan; ion source: ESI-; ion transfer tube temperature: 320°C; ion source heating temperature: 275°C; auxiliary gas: 5-20 Arb; sheath gas: 10-50 Arb; purge gas: 0-3 Arb; SIM mode resolution: 30000; spray voltage: -4000v; scanning conditions were:

[0019]

[0020]

[0021] Preferably, in the above method, in step A, the solution is dissolved in 50% ethanol aqueous solution.

[0022] Wherein, in the above method, in the chromatographic conditions, the injection volume is 1 to 5 μL.

[0023] Preferably, in the above method, under the chromatographic conditions, the injection volume is 1 μL.

[0024] Wherein, in the above method, in the chromatographic conditions, the column temperature is 35-50°C.

[0025] Preferably, in the above method, in the chromatographic conditions, the column temperature is 40°C.

[0026] Preferably, in the above method, in the mass spectrometry conditions, auxiliary gas: 7Arb; sheath gas: 25Arb; sweep gas: 0Arb.

[0027] Wherein, in the above method, in step B, the flavor type of the liquor is strong aroma type, light aroma type, sauce aroma type or rice aroma type.

[0028] Preferably, in the above method, in step B, the concentrations of the 14 higher fatty acids in the mixed standard working solution of the series of concentrations are controlled within 0.005-30 mg / L, and the concentrations of each higher fatty acid in the mixed standard working solution are set to at least six different point values.

[0029] More preferably, in the above method, in step B, the concentrations of the 14 higher fatty acids in the mixed standard working solution of the series of concentrations are 0.005 mg / L, 0.01 mg / L, 0.02 mg / L, 0.05 mg / L, 0.1 mg / L, 0.2 mg / L, 0.5 mg / L, 1 mg / L, 2 mg / L, 5 mg / L, 10 mg / L, 20 mg / L and 30 mg / L.

[0030] Most preferably, in the above method, the linear equation, linear range and linear correlation coefficient of the 14 higher fatty acids are:

[0031]

[0032]

[0033] ; Wherein, x is the concentration value of the target higher fatty acid in the test sample in mg / L; y1 is the peak area of ​​the target higher fatty acid; R 2 is the linear correlation coefficient.

[0034] In the present invention, the concentrations of the ethanol aqueous solution and the formic acid aqueous solution are volume concentrations.

[0035] Beneficial effects of the present invention:

[0036] The present invention utilizes the high resolution capability of ultra-high performance liquid chromatography combined with a quadrupole-electrostatic field orbital trap mass spectrometer in detection and analysis, optimizes the parameters of the target higher fatty acids in the ion source region, selects appropriate parameters according to the best mass spectrum measurement conditions to collect compound mass spectrum data, and realizes accurate quantification of higher fatty acid compounds in liquor by utilizing the accurate mass-to-charge ratio of the target parent ion under direct sampling and selected ion monitoring mode (SIM), reduces the detection time and can well avoid the false positive phenomenon caused by matrix interference, and the sample does not need to undergo cumbersome pre-treatment or derivatization operations, so the operation is simpler. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the standard curve of 14 higher fatty acids in Example 1.

[0038] Figure 2 This is the total ion current diagram of 14 higher fatty acids in the 0.5 mg / L mixed standard solution in Example 1 under SIM mode.

[0039] Figure 3 This is the total ion current diagram of the Luzhou-flavor liquor sample to be tested in Example 1 under SIM mode.

[0040] Figure 4 This is the ion extraction chromatogram of 2-hydroxyoleic acid in the 0.5 mg / L mixed standard solution in Example 1.

[0041] Figure 5 This is the ion extraction chromatogram of 16-hydroxypalmitic acid in the 0.5 mg / L mixed standard solution in Example 1.

[0042] Figure 6 This is the ion extraction chromatogram of arachidonic acid in the 0.5 mg / L mixed standard solution in Example 1.

[0043] Figure 7 This is the ion extraction chromatogram of arachidic acid in the 0.5 mg / L mixed standard solution in Example 1.

[0044] Figure 8 This is the ion extraction chromatogram of myristic acid in the 0.5 mg / L mixed standard solution in Example 1.

[0045] Fig. 9 This is the ion extraction chromatogram of pentadecanoic acid in the 0.5 mg / L mixed standard solution in Example 1.

[0046] Fig.10 This is the ion extraction chromatogram of cis-9-hexadecenoic acid in the 0.5 mg / L mixed standard solution in Example 1.

[0047] Fig.11 This is the ion extraction chromatogram of cis-11-eicosenoic acid in the 0.5 mg / L mixed standard solution in Example 1.

[0048] Fig.12 This is the ion extraction chromatogram of linolenic acid in the 0.5 mg / L mixed standard solution in Example 1.

[0049] Fig.13 This is the ion extraction chromatogram of linoleic acid in the 0.5 mg / L mixed standard solution in Example 1.

[0050] Fig.14 This is the ion extraction chromatogram of stearic acid in the 0.5 mg / L mixed standard solution in Example 1.

[0051] Fig.15 This is the ion extraction chromatogram of oleic acid in the 0.5 mg / L mixed standard solution in Example 1.

[0052] Fig.16 This is the ion extraction chromatogram of lauric acid in the 0.5 mg / L mixed standard solution in Example 1.

[0053] Fig.17 This is the ion extraction chromatogram of palmitic acid in the 0.5 mg / L mixed standard solution in Example 1. DETAILED DESCRIPTION

[0054] The scheme of the present invention will be explained below in conjunction with embodiment.It will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.If no specific technology or conditions are indicated in the embodiment, the technology or conditions described in the document in this area or the product specification are carried out.If the manufacturer is not indicated in the reagents or instruments used, they are all conventional products that can be obtained commercially.Embodiments of the present invention are described in detail below.

[0055] Test Example 1

[0056] (1) Preparation before injection: No pretreatment or derivatization is required. About 1 mL of liquor sample is directly drawn, filtered through a 0.22 μm organic filter membrane, and then injected into the injection vial for testing.

[0057] (2) The LC-MS detection and analysis method includes the following steps: Determine the LC-MS120 analysis parameter conditions as shown in Tables 1, 2, 3 and 4.

[0058] Table 1 Chromatographic conditions

[0059] Modules condition Chromatographic columns Waters ACQUITY UPLC BEH C18(1.7μm,150mm*2.1μm) Mobile phase A: 0.1% formic acid aqueous solution; B: methanol Column temperature 40℃ Injection volume 1μL Flow rate 0.2mL / min

[0060] Table 2 Elution program

[0061]

[0062]

[0063] After comprehensive consideration, the optimal parameters of spray voltage and sheath gas / auxiliary gas / purge gas for each of the 14 fatty acids in Table 5 were obtained, and the mass spectrometry source conditions in Table 3 were obtained.

[0064] Table 3 Mass spectrometry source conditions

[0065] Modules condition Scan Mode SIM scan Ion source ESI- Ion transfer tube temperature 320℃ Ion source heating temperature 275℃ Auxiliary gas 7Arb Sheath Air 25Arb Purge gas 0Arb SIM mode resolution 30000 Spray voltage -4000v

[0066] Table 4 Scanning conditions

[0067]

[0068]

[0069] (3) Optimization results of the ion source area of ​​the higher fatty acid compounds to be tested.

[0070] Table 5 Parameters of the source area of ​​14 higher fatty acid ions at the maximum response of the instrument

[0071] Substance name Spray voltage (v) Sheath gas / auxiliary gas / purge gas (Arb) Linoleic acid -5400 50 / 21 / 0 Myristic acid -5200 40 / 25 / 3 Pentadecanoic acid -5300 40 / 25 / 3 Linolenic acid -3800 10 / 7 / 0 Palmitic acid -3600 10 / 7 / 0 Lauric acid -5500 25 / 5 / 0 Cis-9-hexadecenoic acid -5300 45 / 23 / 2 Stearic acid -4000 40 / 25 / 0 Oleic acid -4000 15 / 5 / 1 Cis-11-eicosenoic acid -4000 20 / 5 / 0 Arachidic acid -4000 20 / 5 / 0 Arachidonic acid -4000 20 / 5 / 0 16-Hydroxypalmitic acid -4000 20 / 5 / 0 2-Hydroxyoleic acid -4500 25 / 5 / 0

[0072] The present invention uses the Optimization option in the MS Tune software in the quadrupole-electrostatic field orbital trap mass spectrometer to optimize the ion source region parameters of the target higher fatty acids, including parameters such as spray voltage, sheath gas, auxiliary gas, and purge gas. By injection sampling, the real-time response of the target higher fatty acids is monitored, and the optimal parameters in Tables 1 to 4 are obtained.

[0073] In the previous experiments, the present invention found that the ethanol degree has an effect on the peak response, so the present invention tested the preparation of standard working solutions with ethanol aqueous solutions and methanol at different concentrations as solvents. Among the ethanol aqueous solutions of different concentrations, the 50% ethanol aqueous solution has basically no effect on the determination of the substance, and methanol also has basically no effect, so direct sampling and detection can be achieved; at the same time, considering that the main components of liquor are ethanol and aqueous solution (the average degree of different types of liquor is about 50°), and most of the higher fatty acids have large differences in response in 50% ethanol aqueous solution and methanol, in order to obtain more accurate quantitative results, the matrix effect (ME) is calculated, and the differences in the standard curves are compared. At the same time, considering the differences in the four flavors of thick and clear rice, the quantitative standard working solution in the present invention is prepared by 50% ethanol aqueous solution to minimize the influence of the solvent on the peak response. The standard curve (y1) of 50% ethanol aqueous solution is shown in Figure 1 and Table 11, the standard curve (y2) of methanol is shown in Table 11.

[0074] Example 1

[0075] A method for simultaneously determining 14 higher fatty acids in Luzhou-flavor liquor comprises the following steps:

[0076] (1) Preparation before injection: Without pretreatment or derivatization, directly aspirate about 1 mL of Luzhou-flavor liquor sample, filter it through a 0.22 μm organic filter membrane, and inject it into the injection vial for testing.

[0077] (2) Sample determination:

[0078] S1. Prepare the stock solution of standard solution: accurately weigh 16-hydroxypalmitic acid, 2-hydroxyoleic acid, arachidic acid, arachidonic acid, cis-11-eicosenoic acid, cis-9-hexadecenoic acid, lauric acid, oleic acid, linoleic acid, linolenic acid, myristic acid, palmitic acid, pentadecanoic acid, and stearic acid, and dissolve them in 50% ethanol aqueous solution to prepare a stock solution of standard solution with a concentration of 200-1000 mg / L.

[0079] S2. Prepare mixed standard working solution: accurately pipette mother liquors of 14 higher fatty acids into a volumetric flask, add 50% ethanol aqueous solution to prepare a mixed standard solution with a concentration of 40 mg / L, and then use 50% ethanol aqueous solution as solvent to dilute step by step to mixed standard working solutions with concentrations of 30, 20, 10, 5, 2, 1, 0.5, 0.2, 0.1, 0.05, 0.02, 0.01, and 0.005 mg / L (taking 0.005 mg / L mixed standard working solution as an example, that is: in this solution, the concentrations of 16-hydroxypalmitic acid, 2-hydroxyoleic acid, arachidic acid, arachidonic acid, cis-11-eicosenoic acid, cis-9-hexadecenoic acid, lauric acid, oleic acid, linoleic acid, linolenic acid, myristic acid, palmitic acid, pentadecanoic acid and stearic acid are all 0.005 mg / L).

[0080] S3. Standard curve drawing: Based on ultra-high performance liquid chromatography combined with quadrupole-electrostatic field orbital trap mass spectrometry to detect the 0.005 mg / L to 30 mg / L mixed standard working solution obtained in S2, the total ion current of 14 higher fatty acids in the mixed standard working solution is shown in the figure below: Figure 2 As shown. TraceFinder software was used to extract and integrate the target ion peaks, and the standard working curve was drawn with the concentration of the target higher fatty acid as the horizontal axis and the peak area as the vertical axis. The standard working curves of 14 higher fatty acids are shown in Figure 1 shown.

[0081] S4, sample determination: Based on ultra-high performance liquid chromatography combined with quadrupole-electrostatic field orbital trap mass spectrometry to detect the sample to be tested in step (1), use TraceFinder software to extract and integrate the target ion peak. Based on the standard working curve drawn in step S3, use the external standard method for quantitative analysis to obtain the content of the non-volatile higher fatty acids in the liquor sample. The total ion current of 14 higher fatty acids in the Luzhou-flavor liquor sample is shown in Figure 2. Figure 3 The results of the contents of 14 higher fatty acids in Luzhou-flavor liquor are shown in Table 6.

[0082] In addition, using this representative Luzhou-flavor liquor as the matrix, 0.005, 1, 5, and 20 mg / L mixed standard solutions were added, respectively, to verify the recovery rates of 14 higher fatty acids added to Luzhou-flavor liquor.

[0083] The data acquisition system used mainly includes liquid chromatography Vanquish Flex Binary connected in series with Orbitrap Exploris 120.

[0084] The liquid phase conditions mainly include the chromatographic column: Waters ACQUITY UPLC BEH C18 (1.7 μm, 150 mm*2.1 μm), mobile phase A is 0.1% formic acid aqueous solution; mobile phase B is methanol. The elution gradient is shown in Table 2. The column temperature is 40°C; the injection volume is 1 ul.

[0085] The mass spectrometry conditions mainly include: the detection mode is electrospray ion source (ESI), the sheath gas is 25Arb, the auxiliary gas is 7Arb, the ion transfer tube temperature is 320°C, the ion source heating temperature is 275°C, the scanning mode is SIM scan, and the resolution is 30000.

[0086] Example 2

[0087] This example provides a method for determining 14 higher fatty acids in light-flavor liquor. The difference from Example 1 is that the sample to be tested is a representative sample of light-flavor liquor and a representative sample of light-flavor liquor to which 0.005, 1, 5, and 20 mg / L of mixed standard solution are added, respectively. The results of the contents of 14 higher fatty acids in light-flavor liquor are shown in Table 6.

[0088] Example 3

[0089] This example provides a method for determining 14 higher fatty acids in Maotai-flavor liquor. The difference from Example 1 is that the sample to be tested is a representative sample of Maotai-flavor liquor and a representative sample of Maotai-flavor liquor to which 0.005, 1, 5, and 20 mg / L of mixed standard solution are added, respectively. The results of the contents of 14 higher fatty acids in Maotai-flavor liquor are shown in Table 6.

[0090] Example 4

[0091] This embodiment provides a method for determining 14 higher fatty acids in rice-flavor liquor. The difference from Example 1 is that the sample to be tested is a representative sample of rice-flavor liquor and a representative sample of rice-flavor liquor to which 0.005, 1, 5, and 20 mg / L of mixed standard solution are added, respectively. The results of the contents of 14 higher fatty acids in rice-flavor liquor are shown in Table 6.

[0092] Results Analysis

[0093] In view of the difficulty of fatty acid cleavage to produce secondary ions, it is impossible to use the MRM mode in triple quadrupole LC-MS for scanning. Therefore, the present invention takes advantage of high-resolution mass spectrometry and only uses primary ions. By optimizing the parameters of the ion source area, the sensitivity of the instrument to the target compound is maximized, and moderate parameters are selected to simultaneously determine 14 higher fatty acid compounds, shorten the scanning window of each target compound, and increase the number of scanning points of different acid compounds to improve the accuracy and reproducibility of quantification. Considering the differences in the matrix in different flavor liquors, it will bring unknown effects on the detection of target substances. In order to more accurately evaluate the accuracy of the invention in determining the above 14 higher fatty acid compounds in liquor, 0.005, 1, 5, and 20 mg / L of mixed standard solutions are added to the representative liquors of the four major flavors of Luzhou-flavor, Light-flavor, Sauce-flavor, and Rice-flavor, and measured as representative samples.

[0094] (1) Finally, the quantitative detection method of Examples 1-4 was used to analyze liquor samples including Luzhou-flavor, Light-flavor, Sauce-flavor, Rice-flavor, etc., and the content (mg / L) of the non-volatile higher fatty acids in the liquor samples was obtained by drawing a standard working curve and using an external standard method. The results are shown in Table 6.

[0095] Table 6 Results of 14 higher fatty acid contents in different flavor liquors (mg / L)

[0096]

[0097]

[0098] In this analysis, through this detection method, the method can quickly and accurately quantify the higher fatty acids in different flavor liquors, including 16-hydroxypalmitic acid, 2-hydroxyoleic acid, arachidic acid, arachidonic acid, cis-11-eicosenoic acid, cis-9-hexadecenoic acid, lauric acid, oleic acid, linoleic acid, linolenic acid, myristic acid, palmitic acid, pentadecanoic acid, and stearic acid. Figure 4 This is the ion extraction chromatogram of the target compound in the 0.5 mg / L mixed standard solution in Example 1, with good chromatographic peak shape and high separation.

[0099] Detection method confirmation

[0100] According to Examples 1-4, 0.005, 1, 5, and 20 mg / L of mixed standard solution were added to representative liquors of four flavor types, namely, strong-flavor, light-flavor, sauce-flavor, and rice-flavor, respectively. Two addition levels were set according to different acid compounds, and each was repeated 5 times. The recovery rate was determined by the above-mentioned analytical method. The specific data results are shown in Tables 7, 8, 9, and 10.

[0101] Table 7 Recovery and precision of 14 higher fatty acids in Luzhou-flavor liquor at different spiked concentrations

[0102]

[0103]

[0104] Table 8 Recovery and precision of 14 higher fatty acids at different spiked concentrations in light-flavor liquor

[0105]

[0106]

[0107] Table 9 Recovery and precision of 14 higher fatty acids at different spiked concentrations in Maotai-flavor liquor

[0108]

[0109]

[0110] Table 10 Recovery and precision of 14 higher fatty acids at different spiked concentrations in rice-flavor liquor

[0111]

[0112]

[0113] According to the addition recovery test, the average recovery rate of different higher fatty acids in Luzhou-flavor liquor is 80%-117%; the average recovery rate in light-flavor liquor is 81%-115%; the average recovery rate in sauce-flavor liquor is 81%-117%; the average recovery rate in rice-flavor liquor is 81%-113%, and the accuracy meets the analysis requirements.

[0114] According to the addition recovery test, the five-repetition intra-day RSD of different higher fatty acids in Luzhou-flavor liquor was 0.4%-7.2%, and the inter-day RSD was 2.4%-9.8%; the five-repetition intra-day RSD in light-flavor liquor was 0.8%-6.8%, and the inter-day RSD was 2.7%-9.7%; the five-repetition intra-day RSD in sauce-flavor liquor was 0.8%-5.2%, and the inter-day RSD was 2.4%-9.6%; the five-repetition intra-day RSD in rice-flavor liquor was 0.9%-8.5%, and the inter-day RSD was 2.6%-9.6%. The precision met the analysis requirements.

[0115] The quantification limit (LOQ) of different higher fatty acids is the concentration of the standard solution when the instrument S / N=10. The quantification limit of different higher fatty acid compounds in liquor is between 2-400ug / L. The method is excellent and can be used to determine the content of trace or trace higher fatty acid compounds in different liquors.

[0116] A standard gradient working curve (y1 is 50% ethanol aqueous solution, y2 is methanol) was formed according to the gradient concentration (30, 20, 10, 5, 2, 1, 0.5, 0.2, 0.1, 0.05, 0.02, 0.01, 0.005 mg / L). The linear correlation coefficient (R 2 ) were all > 0.99, which met the analysis requirements. The specific data results are shown in Table 11.

[0117] Table 11 Linear range, linear equation, linear correlation coefficient and quantification limit of 14 higher fatty acids

[0118]

[0119]

[0120] Note: ME%=[k(y1) / k(y2)-1]*100.

[0121] It should be noted that the specific features, structures, materials or characteristics described in this specification may be combined in any one or more embodiments in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments and features of the different embodiments described in this specification without contradicting each other.

Claims

1. A method for in-situ determination of higher fatty acids in liquor, characterized in that: The following steps are involved: A. Preparation of standard working solution: accurately weigh 16-hydroxypalmitic acid, 2-hydroxyoleic acid, arachidic acid, arachidonic acid, cis-11-eicosenoic acid, cis-9-hexadecenoic acid, lauric acid, oleic acid, linoleic acid, linolenic acid, myristic acid, palmitic acid, pentadecanoic acid and stearic acid, dissolve in 45-55% ethanol aqueous solution to obtain mixed standard working solutions of a series of concentrations; B. Preparation of test samples: directly use liquor as the test sample, or filter the liquor through a 0.22μm organic filter membrane and use it as the test sample; C. Drawing of standard working curves: The mixed standard working solution was injected into an ultra-high performance liquid chromatography combined with a quadrupole-electrostatic field orbital trap mass spectrometer to extract and integrate the target ion peaks. The concentration of the target higher fatty acid was used as the abscissa and the peak area was used as the ordinate to obtain the standard working curves of 14 higher fatty acids respectively; D. Detection of the sample to be tested: inject the sample to be tested into an ultra-high performance liquid chromatography combined with a quadrupole-electrostatic field orbital trap mass spectrometer, extract and integrate the target ion peak under the same detection conditions as in step C, obtain the peak area of ​​the target higher fatty acid in the sample to be tested, and calculate the concentration of 14 higher fatty acids in the sample to be tested according to the standard working curve obtained in step C; The chromatographic conditions are as follows: chromatographic column: Waters ACQUITY UPLC BEH C18, 1.7 μm, 150 mm*2.1 μm; mobile phase: mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: methanol; gradient elution program: ； The mass spectrometry conditions were as follows: scanning mode: SIM scan; ion source: ESI-; ion transfer tube temperature: 320°C; ion source heating temperature: 275°C; auxiliary gas: 5-20 Arb; sheath gas: 10-50 Arb; purge gas: 0-3 Arb; SIM mode resolution: 30000; spray voltage: -4000v; scanning conditions were:

2. The method for in-situ determination of higher fatty acids in liquor according to claim 1, characterized in that: In step A, it is dissolved in 50% ethanol aqueous solution.

3. The method for in-situ determination of higher fatty acids in liquor according to claim 1, characterized in that: In the chromatographic conditions, the injection volume is 1 to 5, preferably 1 μL.

4. The method for in-situ determination of higher fatty acids in liquor according to claim 1, characterized in that: In the chromatographic conditions, the column temperature is 35-50°C, preferably 40°C.

5. The method for in-situ determination of higher fatty acids in liquor according to claim 1, characterized in that: In the mass spectrometry conditions, auxiliary gas: 7Arb; sheath gas: 25Arb; sweep gas: 0Arb.

6. The method for in-situ determination of higher fatty acids in liquor according to claim 1, characterized in that: In step B, the flavor type of the liquor is strong-flavor type, light-flavor type, sauce-flavor type or rice-flavor type.

7. The method for in-situ determination of higher fatty acids in liquor according to claim 1, characterized in that: In step B, in the mixed standard working solution of the series of concentrations, the concentrations of the 14 higher fatty acids are controlled within the range of 0.005 to 30 mg / L, and the concentrations of the higher fatty acids in the mixed standard working solution are set at least at six different point values.

8. The method for in-situ determination of higher fatty acids in liquor according to claim 7, characterized in that: In step B, in the mixed standard working solution of the series of concentrations, the concentrations of the 14 higher fatty acids are 0.005 mg / L, 0.01 mg / L, 0.02 mg / L, 0.05 mg / L, 0.1 mg / L, 0.2 mg / L, 0.5 mg / L, 1 mg / L, 2 mg / L, 5 mg / L, 10 mg / L, 20 mg / L and 30 mg / L.

9. The method for in-situ determination of higher fatty acids in liquor according to claim 8, characterized in that: The linear equations, linear ranges and linear correlation coefficients of 14 higher fatty acids are: Where x is the concentration value of the target higher fatty acid in the test sample in mg / L; y1 is the peak area of ​​the target higher fatty acid; R 2 is the linear correlation coefficient.

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